RT Journal Article
SR Electronic
T1 REP-X: An Evolution-guided Strategy for the Rational Design of Cysteine-less Protein Variants
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 797969
DO 10.1101/797969
A1 Kevin Dalton
A1 Tom Lopez
A1 Vijay Pande
A1 Judith Frydman
YR 2019
UL http://biorxiv.org/content/early/2019/10/08/797969.abstract
AB Site-specific labeling of proteins is often a prerequisite for biophysical and biochemical characterization. Chemical modification of a unique cysteine residue is among the most facile methods for site-specific labeling of proteins. However, many proteins have multiple reactive cysteines, which must be mutated to other residues to enable labeling of unique positions. This trial-and-error process often results in cysteine-free proteins with reduced activity or stability. Herein we describe a general methodology to rationally engineer cysteine-less proteins. Briefly, natural variation across orthologues is exploited to identify suitable cysteine replacements compatible with protein activity and stability. As a proof-of-concept, we recount the successful engineering of a cysteine-less mutant of the group II chaperonin from methanogenic archaeon Methanococcus maripaludis. A webapp, REP-X (Replacement at Endogenous Positions from eXtant sequences), which enables users to design their own cysteine-less protein variants, will make this rational approach widely available.